Growth Performance and Biochemical Analysis of the Genus Spirulina Under Different Physical and Chemical Environmental Factors
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Vol. 10(36), pp. 3614-3624, 3 September, 2015 DOI: 10.5897/AJAR2015.10210 Article Number: EC3B4C055280 African Journal of Agricultural ISSN 1991-637X Copyright ©2015 Research Author(s) retain the copyright of this article http://www.academicjournals.org/AJAR Full Length Research Paper Growth performance and biochemical analysis of the genus Spirulina under different physical and chemical environmental factors Dorothy Kemuma NYABUTO1,2, Kewei CAO1, Alfred Mugambi MARIGA3, Grace Wanjiru KIBUE1,2 , Meilin HE1 and Changhai WANG1* 1College of Resources and Environmental Science, Nanjing Agricultural University, No. 1 Weigang Road, Nanjing 210095, P. R. China. 2Department of Natural Resources, Egerton University, Egerton 536, Kenya. 3College of Food Science and Technology, Nanjing Agricultural University, No. 1 Weigang Road, Nanjing 210095, P. R. China. Received 24 July, 2015; Accepted 21 August, 2015 Spirulina is useful to man in many aspects of life including health, food and cosmetics. In the present study, we aimed at investigating the optimum physical and chemical conditions that promote Spirulina mass production by varying a set of physical and chemical parameters, namely pH levels; Mg2+ ion concentration; nitrogen, phosphorous and carbon sources; salinity and different growing media. Temperature, light intensity, and light/dark cycle were maintained at 30°C±2, 4400 lx and 14:10 respectively throughout the study. At pH 9, the dry weight, protein, and chlorophyll a contents were 7.83±0.29 g/L, 1.34±0.12 mg/mL and 18.64±0.06 µg/mL respectively. At 0.8 mmol/L MgO, maximum dry weight, protein, and chlorophyll a contents were obtained. By using NaNO3 as the source of nitrogen, the dry weight was 2.24±0.13 g/L while the protein and chlorophyll a contents were 3.24±0.30 mg/mL and 2.53±0.24 µg/mL respectively. The highest biomass, chlorophyll a and protein contents were obtained by using K2HPO4 as the source of phosphorous. When NaHCO3 was used as the carbon source, the highest dry weight, protein and chlorophyll a yields were observed. Of the growing media used, Zarrouk’s medium yielded the highest biomass protein and chlorophyll a contents. Furthermore, under different salinities, the optimal dry weight, protein and chlorophyll a yields were obtained at 2.5%. This study provides the basis for high biomass production of Spirulina which is a promising microalga endowed with many health benefits due to its high protein content. Key words: Biomass, chlorophyll a, protein, Spirulina. INTRODUCTION Spirulina platensis is a planktonic photosynthetic populations in tropical and subtropical water bodies that filamentous cyanobacterium that forms massive have high level of carbonate, bicarbonate and pH values *Corresponding author. E-mail: [email protected] Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Nyabuto et al. 3615 Table 1. Composition of Zarrouk’s medium (standard medium, SM). Constituents Composition (g/L) NaHCO3 18.0 NaNO3 2.5 K2HPO4 0.5 K2SO4 1.0 NaCl 1.0 CaCl2.2H2O 0.04 Na2EDTA 0.08 MgSO4.7H2O 0.2 FeSO4.7H2O 0.01 a A5 micronutrient sol. 1 mL/L a A5 micronutrient solution consists of H3BO3, 2.86; MnCl2.4H2O, 1.81; ZnSO4.7H2O, 0.222; CuSO4.5H2O, 0.079; (NH4)6Mo7O24 (g/L). Table 2. Composition of ES medium (West and McBride version). Component Primary stock solution Quantity (1 L) Molar concentration in final medium -4 NaNO3 - 3.85 g 4.53×10 mol/L -5 Na2β-glycerophosphate.5H2O - 0.4 g 1.31×10 mol/L Fe-EDTA solution (see recipe below) 100 mL - Trace metal solution (see recipe below) 20 mL - -7 Thiamine (Vit. B1) 500 mg/L dH2O 8.0 mL 1.19×10 mol/L -8 Biotin (Vit. H) 50 mg/L dH2O 8.0 mL 1.78×10 mol/L -10 Cyanocobalomin (Vit. B12) 25 mg/L dH2O 3.5 mL 6.46×10 mol/L Fe-EDTA solution consists of Na2EDTA.2H2O, 6.00 g/L; Fe (NH4)2(SO4)2.6H2O, 7.00 g/L. The trace metal solution consisted of Na2EDTA.2H2O, 2.548 g/L; H3BO3, 2.240 g/L; MnSO4.4H2O, 0.240 g/L; ZnSO4.4H2O, 0.044 g/L; CoSO4.7H2O, 0.010 g/L. of up to 11. This cyanobacterium features morphological MATERIALS AND METHODS traits of the genus, that is, the arrangement of Microorganism multicellular cylindrical trichomes in an open lift-hand helix along entire length of the filaments (Soni et al., Cyanobacterium Spirulina used in the present study was purchased 2012). The genus Spirulina has gained importance and from Collection Centre of Marine Microalgae, Chinese Academy of international demand for its high phytonutrients Sciences, China. It was grown and maintained in 2 L sterilized value and pigments, which have applications in healthy Erlenmeyer flasks containing 1 L Zarrouk’s medium (Amala et al., foods, feeds, and therapeutics (Becker, 2007). It 2013), chemical composition of which is shown in Table 1, at room temperature, pH 9 with continuous illumination using cool white represents the second most important commercial fluorescent tubes whose light intensity was controlled and microalgae (after Chlorella) for the production of maintained at 2500 lx using the TES-1330A Digital Lux Meter (TES biomass used as healthy food and animal feed Electrical Electronic Corp, ISO 9001:2008). The experiment was (Sotiroudis et al., 2013). Its annual worldwide done in triplicate. Agitation was done thrice a day manually. All the production in the year 2000 was estimated to be reagents used were of analytical grade and were obtained from Shoude Experimental Equipment Co., Ltd, Nanjing. approximately 2000 tonnes (Spolaore et al., 2006). Spirulina has been used as food and nutritional supplements since antiquity (Falquet and Hurni, 2006). It Culture media and growing conditions is generally considered as a rich source of proteins, Zarrouk’s medium (Amala et al., 2013) was used as the standard vitamins, essential amino acids, minerals, essential fatty control medium (SM) and its constituents are shown in Table 1. It acids such as linolenic acid and sulfolipids was autoclaved (LDZX-75KBS Vertical Heating Pressure Steam (Mendes et al., 2003). In addition to poly-unsaturated fatty Sterilizer (Shanghai Shenan Medical Instrument Factory) at 121°C acids, it also has 6 poly unsaturated fatty acids, for 15 min and incubation was done aseptically. ES medium (West phycocyanin and other phytochemicals (Chamorro et al., and McBride Version) (West et al., 1999) was used as the growing medium in cultivation of Spirulina under different salinity levels and 2002). The present study reports the impact of different its constituents are shown in Table 2. The cyanobacterium Spirulina physical and chemical environment for mass production was grown under the following physical conditions: temperature 30 of Spirulina. ±2°C, light intensity 4400 lx and light/dark cycle 14:10. Agitation 3616 Afr. J. Agric. Res. Table 3. Composition of CFTRI medium. Ingredients g/L dH2O NaHCO3 4.5 K2HPO4 0.5 NaNO3 1.5 K2SO4 1.0 NaCl 1.0 MgSO4.7H2O 1.2 CaCl2.2H2O 0.04 FeSO4.7H2O 0.01 Table 4. Composition of Bangladesh media No.3. Ingredients g/L dH2O NaHCO3 2.0 Urea 0.05 NaCl 1.0 Gypsum 1.5 Table 5. Composition of BG-11 (Blue Green-11) medium. Stock solutions g/L dH2O NaNO3 15.0 K2HPO4.3H2O 4.0 MgSO4.7H2O 7.5 CaCl2.2H2O 3.6 Citric acid 0.6 Ferric ammonium citrate 0.6 (Autoclave to dissolve) EDTA 0.1 Na2CO3 2.0 Trace metal mixture 1 ml, g/L H3BO3 2.86 MnCl2.4H2O 1.81 Add each constituent ZnSO .7H O 0.222 separately to ~800 mL of dH2O 4 2 and fully dissolve between Na2MoO4.2H2O 0.39 each addition. Then make up CuSO4.5H2O 0.079 to 1 L Co(NO3)2.6H2O 0.0494 was done thrice a day and the experiments were done in triplicate. 3, 4, 5, 6, 7, and 1, respectively. All the sets of experiment were To achieve growth under different chemical conditions, the done in triplicate. Zarrouk’s medium was set at pH 7, 8, 9 (control), 10, 11 and 12; the MgO concentration was set at 0.0, 0.4, 0.8 (control), 1.2 and 1.6 mmol/L; for nitrogen source 2.5 g/L of NaNO3 (standard), urea and Cultivation NH4Cl were used; for phosphorus source 0.5 g/L of K2HPO4 (standard), KH2PO4 and NaH2PO4 were used and for the carbon Spirulina was cultivated aseptically in the 250 mL sterilized source 18 g/L of NaHCO3 (standard), Na2CO3 and K2CO3 were Erlenmeyer flasks containing 150 mL of respective sterilized media. used; salinities of 1.0, 1.5, 2.0, 2.5, 3.0, 3.5% were used and finally The temperature in the incubator was maintained at 30±2°C and CFTRI media, natural sea water, Bangladesh media No.3, BG-11, illuminated with day-light fluorescent tubes with light intensity at RaO’s, OFERR, and Zarrouk’s media (control) were used as the 4400 lx under Light/dark cycle, 14:10. During the process of growth, different growing media and their constituents are shown in Tables the flasks were shaken manually 3 times per day. The experiments Nyabuto et al. 3617 Table 6. Composition of RaO’s medium. Ingredients g/L dH2O NaHCO3 15.0 K2HPO4 0.5 NaNO3 2.5 K2SO4 0.6 NaCl 0.2 MgSO4.7H2O 0.04 CaCl2.2H2O 0.008 Fe-Ethylene diamine tetra acetate 0.2 Microelement solution 1 mL The microelement solution consisted of H3BO3, 2.86 g/L; MnCl2.4H2O, 1.81 g/L; ZnSO4.7H2O, 0.222 g/L; CuSO4.5H2O, 0.079 g/L; (NH4)Mo7O24, 0.02 g/L.